Efficacy of immunotherapy in ARID1A-mutant solid tumors: a single-center retrospective study

Background Immune checkpoint inhibitors (ICIs), especially those targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1), have introduced a new treatment landscape for many types of tumors. However, they only achieve a limited therapeutic response. Hence, identifying patients who may benefit from ICIs is currently a challenge. Methods 47 tumor patients harboring ARID1A mutations were retrospectively studied. The genomic profiling data through next-generation sequencing (NGS) and relevant clinical information were collected and analyzed. Additionally, bioinformatics analysis of the expression of immune checkpoints and immune cell infiltration levels was conducted in ARID1A-mutant gastric cancer (GC). Results ARID1A mutations frequently co-occur with mutations in DNA damage repair (DDR)-associated genes. Among the 35 ARID1A-mutant patients who received immunotherapy, 27 were evaluable., with the objective response rate (ORR) was 48.15% (13/27), and the disease control rate (DCR) was 92.59% (25/27). Moreover, survival assays revealed that ARID1A-mutant patients had longer median overall survival (mOS) after immunotherapy. In ARID1A-mutated GC patients, receiving ICIs treatment indicated longer progressive-free survival (PFS). Additionally, the incidence of microsatellite instability-high (MSI-H), high tumor mutation burden (TMB-H) and Epstein‒Barr virus (EBV) infection was elevated. Bioinformatic analysis showed significant enrichment of immune response and T cell activation pathway within differentially expressed genes in ARID1A-mutant GC group. Finally, ARID1A mutations status was considered to be highly correlated with the level of tumor infiltrating lymphocytes (TILs) and high expression of immune checkpoints. Conclusions Patients with tumors harboring ARID1A mutations may achieve better clinical outcomes from immunotherapy, especially in GC. ARID1A mutations can lead to genomic instability and reshape the tumor immune microenvironment (TIME), which can be used as a biomarker for immunotherapy. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-01074-1.


Introduction
Immune checkpoint inhibitors (ICIs) have generated clinical efficacy across a wide array of tumor types, including mismatch repair-deficient and microsatellite instability-high (d-MMR/MSI-H) cancers [1,2].However, despite the success of ICIs, the clinical responses varies among patients [3], with only 20-40% of patients benefiting from these revolutionary therapies [4,5].Therefore, exploring novel predictive biomarkers is critical.
AT-rich interaction domain 1A (ARID1A), a gene encoding a large nuclear protein member of the switch/sucrose non-fermentation (SWI/SNF) chromatin remodeling complex, may downregulate corresponding protein levels due to the functional mutation [6].As a known tumor suppressor gene, ARID1A strongly regulates the DNA repair pathway, thus driving tumor formation [7].In our previous study, ARID1A was found to serve as a novel biomarker for the prognosis and sensitivity to ICIs of advanced non-small cell lung cancer (NSCLC) [8,9].This finding indicates that the ARID1A mutation status has potential predictive value in immunotherapy.
In this study, we retrospectively analyzed the genomic alterations and clinical outcomes of patients harboring ARID1A mutations.Then, the predicted functions of ARID1A mutations in gastric cancer (GC) were comprehensively analyzed.

Study population
57 tumor patients from the Affiliated Hospital of Qingdao University were retrospectively studied, including 47 patients with ARID1A mutations and 10 ARID1A wild-type GC patients undergoing immunotherapy.Each patient's baseline information, clinical efficacy and follow-up information were collected.The clinical characteristics are shown in Supplementary Table 1.Treatment response to immunotherapy was evaluated based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.Objective response rate (ORR) was calculated as complete response (CR) rate plus partial response (PR) rate under computed tomography or magnetic resonance imaging.Disease control tare (DCR) was defined as proportion of patients who had a CR, PR, and stable disease (SD) as best overall response during immunotherapy.All procedures were approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.All investigations were carried out according to the rules of the Declaration of Helsinki.

Next-generation sequencing assay
Next-generation sequencing (NGS) assays from Burning Rock Co. and BGI Co. were used to analyze tissue samples.Probe hybridization and high-throughput sequencing were employed to detect the whole-exon region of 310 genes and the hot spot mutation region (including exon, intron or promoter region) of 210 genes.The assay covers single nucleotide variants (SNVs) in the target gene capture exons and short fragment insertion or deletion variants (Indels), copy number variants (CNVs), and gene rearrangements (Rearrangements/Fusions) with breakpoints within the capture range.In tissue specimen mutation detection, using 1% as the minimum threshold for determining the variant allele frequency (VAF) of a mutation.Our analysis focused on genetic mutations in SWI/SNF complex members, including ARID and SMARC family genes, DNA damage response (DDR) genes, immune-related genes, and common cancer driver genes.All variants of unknown significance (VUS) were excluded from further studies, including genes that have not yet approved by clinical practice guidelines or lack clinical evidence.We examined five microsatellite loci (BAT25, BAT26, D17S250, D2S123 and D5S346) from patients' tumor tissue and their matched blood as controls, with 2 or more out of 5 loci mutations regarded as MSI-H, 1 and none mutated loci as microsatellite instability-low (MSI-L)/microsatellite stability (MSS).

Efficacy of immunotherapy in patients with ARID1A mutations
Among the 33 patients receiving ICIs treatment, 27 patients had assessable lesions were evaluable for response.A Waterfall plots were used to show the best observed changes in tumor size (Fig. 2a).The other 6 patients received maintenance therapy with ICIs after surgery, 1 of these patients had disease progression (PD) due to increased cancerous ascites, and none of the other 5 patients had an assessable lesion.Among the evaluable tumors, 13 patients (13/27, 48.15%) achieved a partial response (PR) and complete response (CR), and the DCR was 92.592% (25/27).Totally 7 of 27 were MSI-H, of whom 1 patients achieved a CR, 3 patient achieved a PR, 3 patients had a SD after immunotherapy.
Until the follow-up date, patients receiving immunotherapy achieved a mOS of 51.07 months.In contrast, patients without immunotherapy had a mOS of 41.33 months.Kaplan-Meier survival analysis revealed that the non-treated ICIs group had significantly shorter survival compared to the treated group (Hazard ratio (HR) = 0.346, 95% confidence interval (CI) 0.083-1.446,P = 0.034, Fig. 2b).Additionally, analysis using the cBioPortal database showed no significant difference in OS between ARID1A-altered patients and wild-type (WT) patients (Fig. 2c).However, in a pan-cancer immunotherapy cohort, patients with ARID1A alterations had significantly longer OS, with the difference being statistically significant (P = 6.193e −3 ) (Fig. 2d).

Survival analysis of ARID1A-mutated malignant gastric cancer after immunotherapy
We selected GC patients for further mechanistic analysis.We retrospectively gathered data of 10 ARID1A-WT GC patients who received first-line chemotherapy plus immunotherapy (Supplemental Fig. S1).In addition, 6 GC cohorts from the cBi-oPortal database were analyzed for cancer genomics.ARID1A mutations were detected in 22% (179/855) of GC patients, as shown in Fig. 3a, which indicated that ARID1A mutations was one of the most frequent gene mutations in GC.In terms of the alteration types, truncation mutations were the most common, followed by missense mutations.Survival analysis showed that GC patients with ARID1A mutations had a better prognosis, as demonstrated in Fig. 3b.The disease-free survival (DFS) of ARID1A-altered GC patients was significantly longer than that of ARID1A-WT patients (84.00 months vs. 38.90months, P = 0.0216).Moreover, compared with ARID1A-mutated patients who underwent immunotherapy combined with chemotherapy as first-line treatment, ARID1A mutations was significantly associated with increased PFS (Not reached vs 3.2 months, HR = 0.225, 95% CI 0.061-0.841,P = 0.034, Fig. 3c).
In ARID1A-WT GC from our cohort, CR were observed in 0 patient, PR in 4 patients and SD in 4 patients, 2 patients had progressive disease (PD), resulting in an ORR of 40% and a DCR of 80%.In ARID1A-mutated group, 10 advanced GC patients received chemotherapy in combination with immunotherapy as a first-line treatment, of which 3 cases achieved (30.00%)CR/PR and 7 cases (70.00%) had SD.The ORR and DCR were 30 and 100%, respectively.The difference in ORR  between two group was statistically insignificant (40 vs. 30%, respectively, P = 0.1382, Fig. 3d).However, the difference in DCR showed statistical significance (80 vs. 100%, respectively, P < 0.0001, Fig. 3e).

The clinical attributes in ARID1A-mutated GC patients
We assessed the correlation between ARID1A status and clinical attributes in GC, considering the extensive influence on immunotherapy responsiveness.In our cohort, TMB value was significantly higher in GC patients with ARID1A mutations (Wilcoxon test P = 0.0088, Fig. 4a).In addition, more GC patients in the mutant group were identified with MSI-H, which suggested that these patients had more genomic instability (Fisher's exact test P < 0.0001, Fig. 4b).The same results were corroborated in the cBioPortal database (Fig. 4c).Interestingly, the proportion of EBV-positive GC patients with ARID1A mutations was also higher than that of ARID1A-WT patients (Wilcoxon test P = 0.06, Fig. 4d).

Correlations between ARID1A mutations and tumor immune microenvironment (TIME)
The relationship between ARID1A and TIME was further studied.A total of 904 DEGs, including 259 upregulated and 645 downregulated genes, were subjected to functional analysis via the DAVID website.GO term analysis revealed the accumulation of DEGs in T-lymphocyte activation, immune response, and inflammatory response.Regarding molecular function, DEGs were involved in receptor binding (Fig. 5a).KEGG pathway analysis showed that the DEGs were enriched in antigen processing and presentation pathways and cell adhesion molecules (Fig. 5b).According to the DisGeNET analysis, DEGs were found to be significantly enriched in colorectal tumors, liver cancer, stomach cancer, inflammation, etc. (Fig. 5c).Furthermore, the DEGs may be involved in Epstein-Barr virus (EBV) infection, the Wnt signaling pathway, the chemokine signaling pathway and the Hippo signaling pathway.Moreover, the correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and ARID1A mutations status was analyzed in GC.Based on the TISIDB databases, we found a correlation between ARID1A mutations and the abundance of infiltrating immune cells, including activated CD8 + T cells (P = 2.52e −6 ), activated CD4 + T cells (P = 3.8e −11 ), activated dendritic cells (P = 0.000443), natural killer cells (P = 0.017), Th1 cells (P = 0.0437) and Th2 cells (P = 0.00287) (Fig. 6a).The association of ARID1A mutations with immune checkpoints was also analyzed.The results displayed that ARID1A mutations in GC was significantly associated with CD274 (P = 7e −6 ), PDCD1 (P = 0.0344), TIGIT (P = 0.00202), CTLA4 (P = 0.00873), HAVCR2 (P = 0.00053), and LAG3 (P = 1.82e −5 ) (Fig. 6b).

Discussion
The failure of an adequate immune response to standard immunotherapy is a major clinical challenge [18].Some elegant studies have described ARID1A mutations may be closely related to tumor immunity and immunotherapy [19][20][21].However, most of these studies were basic research.A retrospective clinical study from real-world is lacking, and genomic features of the ARID1A-mutated patients remains unclear.
In our study, immunotherapy significantly prolonged the survival time of patients with ARID1A mutations.Among these patients, 2 patient achieved CR, and 11 patients achieved PR.All but PD patients had varying degrees of tumor shrinkage.Moreover, we validated our results on survival benefits in an independent immune cohort from a database.Thus, identifying potential immune therapeutic options based on ARID1A alterations is promising.
In line with other reports [22], the majority of ARID1A mutations in our study were inactivating mutations that could result in the loss of ARID1A expression.Moreover, in our cohort, ARID1A mutations are often accompanied by co-mutations in genes involved in DDR pathways, including ATM, ATR , BRCA2, etc.We found MMR pathway genes were frequently mutated, with MSH6 mutations being the most common.These critical genes alterations decrease DNA repair capacity, therapy increasing tumor burden and activating immunity.Interestingly, in all patients, 48.94% patients had high TMB, and 27.66% patients were microsatellite instable.Consistent with the cBioPortal database Fig. 4 Comparison of the clinical attributes of the ARID1A wild-type and mutated GC groups.a. ARID1A mutations is associated with TMB level in GC of our cohort.b.The associated between MSI status and ARID1A mutations c.Comparison of TMB and MSI between ARID1A wild-type and mutated groups of the cBioPortal database.d.The correlation of EBV infection with ARID1A mutations.GC Gastric cancer, TMB Tumor mutation burden, MSI Microsatellite instability, EBV Epstein-Barr virus analysis results, patients harboring ARID1A mutations were more likely to have increased genomic instability, while TMB levels were also significantly elevated.Currently, MSI-H/dMMR and TMB-H have already been recognized as positive indications for immunotherapy [23].Thus, ARID1A mutations have potential application value for the prediction of response to ICIs.Shen et al. revealed that ARID1A inactivation hampered the recruitment of MSH2 to chromatin during DNA replication and induced dMMR in a proteomic screen [20].In addition, deletions in ARID1A expression were associated with methylation of the promoter of the MLH1 gene [24].These could be underlying mechanism.Furthermore, the DCR in ICIs-treated patients with MSS was 88.24% (15/17).This finding suggests that ARID1A mutations are still an effective predictor of ICI efficacy regardless of MSI status.
Previous researchers have revealed that EBV-positive tumors are related to checkpoint blockade responses [25,26].A possible factor is that EBV-positive cancers often exhibit amplification of the 9p24.1 locus linked to the overexpression of JAK2, CD274, and PDCD1LG2 [27].Our findings showed that ARID1A mutations was associated with EBV infection in GC.This association implied potential benefits for ICIs therapy in some patients with ARID1A mutations in GC.Unfortunately, GC patients in our cohort were not tested for EBV status.In pan-cancer patients, we investigated the correlation between ARID1A mutations and the immune cell microenvironment in GC.The latest literature [28] showed that, compared to HER2-positive GC patients, ARID1A mutations was significantly enriched in HER2-negative GC patients.HER2-negative GC with ARID1A mutations may be sensitive to ICIs, due to increased T-cell lymphocytosis.TILs play a vital role in the improved survival of cancers, and both the quantity and quality of TILs are possible factors in determining immune therapeutic benefits, especially T lymphocytes [29].Moreover, NK cells were proven to be critical for the therapeutic effects of PD1 blockade [30].Our findings confirmed that ARID1A mutations are associated with immune infiltration and immunosuppressive receptors in the TIME.GC patients harboring ARID1A alterations had a greater abundance of activated CD8 + /CD4 + T lymphocytes, DCs, and NK cells, which improved the sensitivity to ICIs.Furthermore, some immunosuppressive targets, such as PD-L1, CTLA-4, HAVCR2, LAG3, and TIGIT, had a significant expression difference in ARID1A-mutant GC.At present, accumulating studies have shown that HAVCR2 and LAG3 are valuable as potential targets for immunotherapy, and preclinical tumor models have shown that the use of immunoinhibitors to block HAVCR2, LAG3, and TIGIT restricts the growth of cancer masses [31][32][33].Admittedly, our study has some limitations.This work is a single-center retrospective study, and multicenter prospective studies are needed to further verify the conclusions.

Conclusions
Based on a single-center retrospective analysis, we concluded that ARID1A mutations were often co-mutated with DNA damage response genes and could be associated with better immunotherapy outcomes in solid tumors.Bioinformatics analysis confirmed the elevated expression of immunosuppressive receptors and increased immune cell infiltration in ARID1A-mutant tumors, both of which are beneficial to the use of ICIs and affect patient outcomes.Therefore, ARID1A can serve as a novel biomarker for immunotherapy for malignant tumors, especially GC.

Fig. 1
Fig. 1 Molecular characteristics of patients with ARID1A mutations.Blue boxes indicate missense mutations, orange boxes are truncating mutations, yellow boxes present copy number gain, purple boxes are splice mutations, black boxes are deep deletion, red boxes are fusion, and green boxes are MSI-H, brown boxes are MSS/MSI-L, and gray boxes present unknow.NA Not applicable, MSI Microsatellite instability, MSS Microsatellite stability, PD-L1 Programmed cell death ligand-1

Fig. 2 Fig. 3 ARID1A
Fig. 2 Patients harboring ARID1A mutations have better clinical outcomes after immunotherapy.a Waterfall plot of the maximum shrinkage rate of measurable targeted lesions compared with baseline b.Patients with ARID1A mutations who received immunotherapy exhibited longer OS in our cohort (P = 0.034).c.ARID1A mutations status was not correlated with OS in pan-cancer setting by cBioPortal database analysis d. OS among patients received immunotherapy with ARID1A mutations or not from the cBioPortal database.OS Overall survival

Fig. 6
Fig. 6 Correlations between ARID1A mutations and tumor immune environment in GC. a ARID1A mutations was highly associated with immune cells infiltration.b.The expression of immune checkpoint was influenced by ARID1A mutations status.GC Gastric cancer